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Background: Lutein and its isomerzeaxanthin are safe natural compounds. They are able to reduce the development of tumor and other chronic diseases. The objectives of the current study was to examine the cytotoxicity of lutein isolated and purified from alfalfa, safe and low-cost plant, on five different human cancer cell lines, namely (MCF-7), (HepG2), (A549), (PC3), and (HCT116), as well as normal (HFB4) cells. Method: We examined the cytotoxicity of lutein purified from Medicago sativa L and evaluate its activity against human liver HepG2, breast MCF-7, lung A549, prostate PC3, and colon HCT116 cancer cell lines using SRB assay in comparison with doxorubicin as a reference drug. Results: Results revealed that the tested extract could be a more promising anticancer agent in the case of MCF-7 (IC50, 3.10±0.47 µg/ml) compared with standard drug doxorubicin (IC50, 2.90±0.30 µg/ml). Moreover, the extract showed a moderate effect on HepG2 (IC50, 6.11±0.84 µg/ml) versus doxorubicin (IC50, 2.90±0.30 µg/ml); meanwhile, the extract showed no activity against A549, PC3, and HCT116 cells. The results further revealed that the extract had no toxicity against the growth of normal HFB4 cells versus doxorubicin. Conclusion: Lutein-rich extract from alfalfa had a major antiproliferative role in breast MCF-7 and liver HepG2 compared to doxorubicin. [ABSTRACT FROM AUTHOR]